The multicatalytic proteinase complex (MPC), also referred to as the proteasome, is an unusually high molecular weight proteinase (about 700 kDa, 19S), that is found in both the cytoplasm and nucleus of a wide variety of eukaryotic cell types. The MPC is composed of about 28 low molecular weight subunits (21,000-34,000 daltons) of which 13-15 are non-identical. See, e.g., Orlowski, M., Biochemistry 29:10289-10297 (1990) and Orlowski, M., J. Lab. & Clin. Med. 121:187-189 (1993) for reviews.
The subunits of the MPC are organized in four stacked rings, each composed of 6-7 subunits surrounding a central water-filled canal. The MPC is highly conserved evolutionarily, having been found in all eukaryotic cells studied, and may constitute up to 0.5-1.0% of the total protein in tissue homogenates. The MPC has been found in both the cytoplasm and nucleus of cells, suggesting a functional role in both of these compartments. (Tanaka et al., J. Cell Physiol. 139:34-41 (1989); Amsterdam et al., Proc. Natl. Acad. Sci., USA 90:99-103 (1993)). A variety of evidence suggests that the MPC is essential for cell viability and proliferation, that it is involved in antigen processing, degradation of nuclear oncoproteins and control of the mitotic cycle through degradation of cyclins.
Adding to its importance in regulating a variety of biological effects is the finding that the MPC is conserved across the evolutionary spectrum, as indicated by immunological cross-reactivity of complexes from eukaryotic species as diverse as yeast, Drosophila and mammalian species. Moreover, a related, but simpler, protein composed of only two subunits has been found in archaebacteria (see Dahlmann et al., FEBS Lett. 251:125-131 (1989)).
The MPC is believed to constitute the catalytic core of a large 26S multisubunit cytoplasmic particle, apparently required inter alia for the ubiquitin-dependent and ubiquitin-independent pathways of intracellular proteolysis (Eytan et al., Proc. Natl. Acad. Sci. USA 86:7751-7755 (1989); Rechsteiner et al., J. Biol. Chem. 268:6065-6068 (1993)). These pathways are believed to be involved in the degradation of cyclins and other short-lived proteins such as nuclear oncogene products (Glotzer et al., Nature (London) 349:132-138 (1991)); Ciechanover et al., Proc. Natl. Acad. Sci. USA 88:139-143 (1991)) and ornithine decarboxylase (Murakami et al., Nature (London) 360:597-599 (1992)). These findings suggest a role of the MPC in regulating cell growth and division (mitosis). Additional studies demonstrated a change in the distribution and activity of the MPC with stages in the cell cycle and that disruption in any one of 12 out of 13 genes encoding yeast MPC subunits results in an arrest in cellular proliferation or an inability to degrade proteins, also suggesting a role for the MPC in cellular growth and division (Fujiwara et al., J. Biol Chem. 265:16604-16613 (1990); Beynon, Int. Committee on Proteolysis News Letter, January, 1-2 (1994)).
The MPC also appears to be required for proper antigen processing by cells of the immune system, based on observations that low molecular weight peptides ("LMPs"), which are structurally similar (homologous) to MPC subunits, are precipitated by antibodies to class I major histocompatibility (MHC) molecules (Brown et al., Nature (London) 353:355-360 (1991)). Moreover, genes for LMPs have been mapped to the class II region of the MHC (Goldberg et al., Nature (London) 357:375-379 (1992)). In addition, MPC and LMP have similar subunit patterns and, as noted, serologically cross-react. All of these observations indicate that the MPC may be responsible for processing and presentation of MHC class I antigens, which are involved in numerous immunological functions and reactions.
While much is known about the biochemistry and functions of the MPC, there is still much that remains unanswered. For example, questions such as how many of the subunits are proteolytically active and what activities are expressed by different subunits have not been answered. Dissociation of the complex results in total loss of proteolytic activity, and all attempts to isolate active subunits have resulted in failure. The integrity of the intact proteasome appears to be required for biological activity. Thus, studies on the specificity of the various MPC components require using the intact, native complex.
Early studies of the MPC led to the delineation of three different proteolytic activities, each associated with a distinct component of the complex (Wilk and Orlowski, J. Neurochem. 35:1172-1182 (1980); Wilk and Orlowski, J. Neurochem. 40:842-849 (1983); Orlowski and Wilk, Biochem. Biophys. Res. Comm. 101:814-822 (1981)). These three activities have been classified, on the basis of the amino acid residues in the P.sub.1 position of the cleaved substrate, as (1) trypsin-like (T-L; cleavage on the carboxyl side of basic residues), (2) chymotrypsin-like (ChT-L; cleavage on the carboxyl side of hydrophobic residues) and (3) peptidylglutamyl-peptide hydrolyzing (PGPH; cleavage on the carboxyl side of glutamyl residues). All three components possess activity toward peptidyl-arylamide bonds. Each of these three activities is inactivated by 3,4-dichloroisocoumarin (DCI), which is known to react with serine residues in the active site of proteases (Orlowski et al., Biochemistry 28:9270-9278 (1989)).
Recent studies have elucidated two additional proteolytic components of the MPC, one which cleaves preferentially on the carboxyl side of branched chain amino acids, designated (BrAAP), and the other, designated SNAAP, which cleaves on the carboxyl side of the small neutral amino acids, alanine and glycine (designated SNAAP) (Orlowski et al., Biochemistry 32:1563-1572 (1993)). Neither the BrAAP nor SNAAP components are capable of cleaving peptidyl-arylamide bonds. Moreover, the BrAAP component is greatly activated upon exposure of the MPC to DCI.
A significant body of evidence indicates that the BrAAP component represents a major factor in protein degradation and that this component may be responsible for most of the protein-degrading activity of the MPC. Thus, the identification and elucidation of the BrAAP component substrate specificity and design of synthetic inhibitors of the MPC (BrAAP component) has use in inhibiting intracellular protein degradation, MHC-mediated immunological reactivities, as well as effecting inhibition of intracellular proteolysis and inhibition of cellular growth and division.